Synthesis and biological evaluation of novel thiocolchicine-podophyllotoxin conjugates

Article abstract of DOI:10.1016/j.ejmech.2009.09.047

The synthesis and biological evaluation of 9 dimeric compounds obtained by condensation of thiocolchicine and/or podophyllotoxin with 6 different dicarboxylic acids is described. In particular, tubulin assembly assay and immunofluorescence analysis results are reported. The biological data highlighted three compounds as being more active than the others, having a marked ability to inhibit the polymerization of tubulin in vitro and causing significant disruption to the microtubule network in vivo. The spacer unit was found to have a significant effect on biological activity, reinforcing the importance of the design of conjugate compounds to create new biologically active molecules in which the spacer could be useful to improve the solubility and to modulate the efficacy of well known anticancer drugs.

Full text of DOI:10.1016/j.ejmech.2009.09.047

European Journal of Medicinal Chemistry 45 (2010) 219–226
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of novel thiocolchicine–
podophyllotoxin conjugates
,
a
b
c
c
Daniele Passarella a , Bruno Peretto , Raul Blasco y Yepes , Graziella Cappelletti , Daniele Cartelli ,
*
Cristina Ronchi d, John Snaith e, Gabriele Fontana f, Bruno Danieli a, Jurgen Borlak g
a
`
Dipartimento di Chimica Organica e Industriale, Via Venezian 21, Universita degli Studi di Milano, 20133 Milano, Italy
b Centro de Investigacion Principe Felipe, Avda. Autopista del Saler 16, Valencia, Spain
c
`
Dipartimento di Biologia, Universita degli Studi di Milano, Via Celoria 26, Milano, Italy
d
`
D.I.P.A.V., Sezione di Biochimica, Universita degli Studi di Milano, Via Celoria 10, Milano, Italy
e School of Chemistry, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK
f Indena S.p.a., Via Ortles 19, Milano, Italy
g Fraunhofer Institute of Toxicology and Experimental Medicine, Medical School of Hannover, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and biological evaluation of 9 dimeric compounds obtained by condensation of thio-
colchicine and/or podophyllotoxin with 6 different dicarboxylic acids is described. In particular, tubulin
assembly assay and immunofluorescence analysis results are reported. The biological data highlighted
three compounds as being more active than the others, having a marked ability to inhibit the poly-
merization of tubulin in vitro and causing significant disruption to the microtubule network in vivo. The
spacer unit was found to have a significant effect on biological activity, reinforcing the importance of the
design of conjugate compounds to create new biologically active molecules in which the spacer could be
useful to improve the solubility and to modulate the efficacy of well known anticancer drugs.
Ó 2009 Elsevier Masson SAS. All rights reserved.
Received 26 June 2009
Received in revised form
23 September 2009
Accepted 28 September 2009
Available online 6 October 2009
Keywords:
Thiocolchicine
Podophyllotoxin
Tubulin polymerization
Microtubule network
Anticancer drugs
1. Introduction
in partial unfolding of the secondary structure of
b-tubulin at the
carboxy terminus and prevents polymerization. [8]
Microtubules are the key components of the mitotic spindle of
eukaryotic cells and they play an important role in cell division. The
discovery of natural and synthetic substances capable of interfering
with the polymerization or depolymerization of microtubules has
gained much attention because microtubules are an attractive
pharmacological target for the design of anticancer drugs. Indeed,
a large number of tubulin binding agents are in clinical develop-
ment, and the recent finding that some tubulin binding agents
target the vascular system of tumors (antiangiogenic activity) has
extended their use still further. [1–4] There is conclusive evidence
that microtubule disruption causes cell cycle arrest mainly at the
G2/M phase, and subsequently programmed cell death. In this
regard, thiocolchicine 1 [5] and podophyllotoxin 2 [6] behave in the
same way as colchicine 3 [7] (Fig. 1): their binding to tubulin results
The importance of tubulin in cell replication, the promising
application of antiangiogenic therapies and the recent discovery
that antiangiogenic agents can normalize abnormal tumor vascu-
lature, leading to more efficient penetration of chemotherapeutic
drugs [9], justify the need for new tubulin-targeted compounds
[10]. In particular, there is a need for compounds with increased
selectivity to improve the antitumor activity and to reduce toxicity,
which is often severe [11].
Various strategies are being developed to increase the effects of
different antitubulin drugs. [12] We have elected to exploit
bifunctional small molecules [13], an idea based on the concept of
multivalency [14], the dominant interaction in biological systems.
Multivalency is now accepted as an effective strategy for designing
ligands, inhibitors and drugs, and it has proved useful in enhancing
the activity and selectivity of some monomeric leads by forming
divalent homo- and heterodimers. [15] As part of our research on
the synthesis of new potential antitumor compounds [16], we
reported previously the synthesis of taxoid–thiocolchicine hybrids
* Corresponding author. Tel.: þ02 50314080; fax: þ02 50314078.
E-mail address: Daniele.Passarella@unimi.it (D. Passarella).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.09.047

220
D. Passarella et al. / European Journal of Medicinal Chemistry 45 (2010) 219–226
OH
O
O
MeO
HO
S
S
S
HO
O
O
O
H
N
H
S
S
S
OH
OH
O
MeO
MeO
O
O
O
4
7
O
O
O
NHBoc
OH
MeO
OMe
R
OH
S
OMe
HO
HO
S
O
NHBoc
O
1
R = SMe
R = OMe
2
5
8
3
O
O
NHBoc
Fig. 1. Cochicine, thiocolchicine and podophyllotoxin.
HO
OH
OH
HO
O
NHBoc
O
[17], and more recently we have prepared binary adducts of Vinca
alkaloids with podophyllotoxin, thiocolchicine and baccatin III [18].
We have also approached the problem by a target-assisted dynamic
combinatorial library of thiocolchicine–podophyllotoxin deriva-
tives based on the disulfide bond exchange reaction [19]. Biological
evaluations revealed that some of the compounds that we prepared
behave as multifunctional tubulin inhibitors and could be inter-
esting probes to investigate not only tubulin biology but also the
complex, and so far poorly understood, relationship between
tubulin and cytotoxicity. In addition, quite surprising biological
properties were displayed, probably due to the interaction with
targets other than tubulin. These results encouraged us to continue
our program directed towards the synthesis of hybrids of naturally
occurring antimitotic products.
6
9
Fig. 2. The dicarboxylic acids used as linkers.
O
O
NHBoc
OBn
BnO
BnO
NHBoc
NHBoc
O
11
10
O
NHBoc
Here we report the synthesis of six new conjugates of thio-
colchicine and podophyllotoxin, and their detailed biological eval-
uation along with three other conjugates that we have previously
prepared [19].
OH
HO
NHBoc
O
9
Scheme 1. Preparation of the linker 9.
2. Results and discussion
Dicarboxylic acid 9 was synthesised from L-N-Boc-allylglycine.
Protection of the carboxylic acid as a benzyl ester (Scheme 1)
provided compound 10 that was submitted to a cross-metathesis
reaction using Grubbs’ 2nd generation catalyst, giving a mixture of
E and Z isomers of 11 in 64% yield. Similar yields were obtained
when microwaves were used in place of conventional heating.
Hydrogenolysis of the benzyl esters and reduction of the double
bond was accomplished by catalytic hydrogenation (Pd/C) using
ethyl acetate as solvent. There was no evidence in the NMR spectra
of epimerization during this synthetic sequence.
2.1. Chemistry
Six different dicarboxylic acids, four of which contain a disulfide
bond, (Fig. 2) were used as the linker molecules to form the dimers.
The dicarboxylic acids were: dithiodiglycolic acid 4; dithiodipro-
pionic acid 5; dithiodibutyric acid 6; adipic acid 7; N-Boc-cystine
8; and 2,7-bis(tert-butoxycarbonylamino)octanedioic acid 9. It was
hoped that the amino groups in the latter two dicarboxylic acids
would enhance the water solubility of the target dimers.
O
O
O
O
YH
Route A
X
X
X X
HO
OH
Y
Y
R
R
R
R
1.
2.
YH
YH
Route B
O
O
X
X
Y
Y
O
O
R
R
X
X
Y
Y
Route C
X = S
R
R
Scheme 2. The three routes used for homo- and heterodimer synthesis.

D. Passarella et al. / European Journal of Medicinal Chemistry 45 (2010) 219–226
OMe
221
MeO
O
O
R
R
O
O
H
N
H
H
S
n
S
N
MeO
MeO
OMe
OMe
H
n
O
O
H
H
O
O
O
O
MeO
MeO
OMe
OMe
O
O
O
O
SMe
SMe
O
O
12 R = H, n = 0
13 R = H, n = 1
OMe
O
OMe
19
14 R = NH2, n = 1
O
MeO
OMe
OMe
R
R
n
H
H
N
H
H
N
H
H
N
MeO
MeO
OMe
OMe
O
OMe
OMe
n
S
S
H
n
n
O
O
O
O
MeO
MeO
O
O
O
O
O
SMe
SMe
SMe
OMe
15 R = NH2, n = 1
16 R = H, n = 0
20 n = 3
O
O
O
MeO
MeO
O
O
O
R
R
n
R
R
H
H
O
N
S
n
S
O
S
n
S
O
H
n
H
H
MeO
O
O
OMe
OMe
O
O
MeO
MeO
OMe
OMe
O
O
O
O
O
O
O
SMe
OMe
OMe
OMe
17 R = H, n = 0
18 R = NH2, n = 1
21 R = NHBoc, n = 1
Fig. 3. The homo- and heterodimers.
The synthesis of the homo- and heterodimers was accomplished
according to Scheme 2. Route A shows the preparation of a homo-
dimer by a simple coupling reaction; route B illustrates the prep-
aration of a heterodimer via sequential coupling reactions; route C,
applicable when the linker contains a disulfide bond, shows the
preparation of a heterodimer by disufide exchange between two
homodimers.
between the corresponding Boc-protected derivatives of 14 and 18,
according to the method we have previously reported [19], but
unfortunately the product of Boc-group removal proved too
unstable for characterisation.
2.2. Biological evaluation
The formation of the homodimers 12–19 (Fig. 3) was straight-
forwardly achieved by condensation of the dicarboxylic acids with
podophyllotoxin or deacetylthiocolchicine, mediated by DCC and
DMAP. The removal of the tert-butoxycarbonyl group from the
compounds obtained by condensation with N-Boc-cystine 8 and
2,7-bis(tert-butoxycarbonylamino)octanedioic 9 acid gave rise to
homodimers with an improved water solubility (1.2–1.5 mg/ml as
hydrochloride salts). Heterodimer 20 was obtained by a two-step
condensation reaction. First, deacetylthiocolchicine was reacted
with 4,40-dithiodibutyric 6 acid to give the monoamide thio-
colchicine derivative that was subsequently reacted with podo-
phyllotoxin, affording 20 (Fig. 3) in an overall 56% yield. The
heterodimer 21 [20] (Fig. 3) was prepared by disulfide exchange
2.2.1. Cytotoxicity by MTS assay
All of the compounds displayed moderate cytotoxicity across
a broad panel of cell lines: A549 (human lung carcinoma), Caco-2
(Human Caucasian colon adenocarcinoma), HepG2 (Human
Caucasian hepatocyte carcinoma), KLN205 (Mouse squamous cell
carcinoma) and eleven transformed mouse cell lines from lung
tumors of double c-myc and c-raf transgenic mice (A2C12, yB8,
yD12, bD10, yA7, yA3, B3, bD5, A2B1, yD1, Craf/Cmyc). Notably, the
latter cell lines display epigenetic plasticity and express several
tumour stem cell markers providing a mechanistic insight into lung
cancer as recently reported by us [21]. In the human cell lines the
IC50 values were in the range 0.5–1
the murine cell lines were more resistant, with IC50 values typically
in the region of 10 M.
mM after 24 h incubation, whilst
m
Table 1
2.2.2. Tubulin assembly assay
Tubulin (3 mg/ml) was polymerized in the absence or presence of 10 mM solution of
To get an insight into the potential biological activity of the
dimers we investigated their ability to affect tubulin polymeriza-
tion in vitro. In order to assess the effect of the compounds on
tubulin assembly, bovine tubulin (purified from brain) was mixed
with a standard solution of each sample in the absence of GTP.
selected compounds and ratio of polymerized/unpolymerized tubulin reported.
P/U (mean ꢀ SEM)
Control
1
2
1.82 ꢀ 0.06
0.87 ꢀ 0.14*
0.80 ꢀ 0.11*
0.74 ꢀ 0.17*
0.98 ꢀ 0.04*
1.13 ꢀ 0.23*
1.83 ꢀ 0.45
0.75 ꢀ 0.16*
1.51 ꢀ 0.21
1.29 ꢀ 0.29
1.20 ꢀ 0.10
1.36 ꢀ 0.29*
12
13
14
15
16
17
18
19
20
Table 2
The novel thiocolchicine–podophyllotoxin conjugates were grouped on the grounds
of the severity of their anti-microtubule action in A549 cells.
Group
Effect (severity of the action)
Compounds
I
mild effect
15, 19
II
III
50% of the cells become roundish
network completely disappears
14, 16, 17
1, 2, 12, 13, 18, 20
*P < 0.05 vs. control, according to ANOVA, Dunnet post-hoc.

222
D. Passarella et al. / European Journal of Medicinal Chemistry 45 (2010) 219–226
Fig. 4. Microtubule organization in human lung carcinoma cell line A549 exposed for 1 h and 24 h to solvent vehicle alone (control) or 0.5
mM selected compounds belonging to
group I, group II (compound 16), and group III, as revealed by immunofluorescence localization of -tubulin (red) and nuclei staining (blue). Bar: 10
a
m
m.
The solutions were then incubated at 37 ꢁC to allow slow binding
drugs to bind to the tubulin, and after 15 min GTP was added to
initiate microtubule assembly. After 30 min the polymerized and
the unpolymerized fractions were separated by centrifugation and
quantified by densitometry. Reported in Table 1 are the ratios of
polymerized/unpolymerized tubulin obtained in the presence of
the different compounds.
It can be seen that the best inhibitors of tubulin polymerization
are the homodimers of thiocolchicine, with 12, 13 and 16 showing
comparable levels of activity to the parent compound, while 14
shows slightly reduced activity. Compounds 17, 18, 19 and 20
incorporating podophyllotoxin, either as homodimers or as heter-
odimers with thiocolchicine, are all rather less active than either
parent monomer. Interestingly, dimers built up with the same
moieties connected by different spacers showed different effects on
tubulin polymerisation. Amino substitution on the linker
(compounds 14, 15, 18), whilst improving aqueous solubility, did
not improve the activity, and may even have contributed to
a decrease in the ability to inhibit tubulin polymerization.
heavy fragmentation of the microtubule network in the perinuclear
region. Particularly noteworthy is compound 16 that induces the
peculiar fragmentation and reorganization of microtubules into
short bundles. Group III includes the moieties thiocolchicine and
podophyllotoxin, along with 12, 13, 18 and 20. These compounds
induce the most severe effect on microtubular organization in A549
cells: the network completely disappears at early time points;
almost all of the cells become roundish following a treatment of
24 h and they show evident apoptosis, as indicated by the frag-
mented nuclei.
2.2.4. In vitro activity with L-glutathione
The biological data highlighted compounds 12, 13 and 16 as the
most active. Compounds 12 and 16 differ only in the fact that the
disulfide bond of 12 is replaced by a two-carbon chain in 16, yet
there was a noticeable difference in their in vivo activity; it is
possible that their rather different behavior in the modification of
the cytoskeleton network (with 16 in Group II and 12 in Group III,
Fig. 3) could be due to cleavage of the disulfide bond in 12, some-
thing that clearly cannot occur in 16. Disulfide cleavage could be the
consequence of the high levels of glutathione (GSH) present in
tumour cells. To determine whether 12 could undergo disulfide
cleavage, we evaluated the action of glutathione on compound 12
in aqueous solution. After an incubation of 3 h we detected the
organic species in solution by high resolution ESI-MS. We
confirmed the effective reductive cleavage of the disulfide bond by
detection of the disulfide derivative of glutathione (GSH) with
sulfahydrylthiocolchicine (Thio–CO–CH2–SH).
2.2.3. Immunofluorescence analysis
In order to confirm the anti-microtubulule effect in cells we
investigated microtubule structure and distribution in human lung
carcinoma cell line A549 exposed to the dimers by indirect
immunofluorescence using anti
A549 cells were incubated for 1 h and 24 h in the presence of the
different compounds (final concentration 0.5 M). We put the
a-tubulin antibodies.
m
compounds into three categories as a function of the severity of
action on microtubule organization in A549 cells (Table 2), and
Fig. 4 shows representative microtubule organization for each
category.
3. Conclusions
In control cells, we observed a widespread network of long
microtubules other than the typical accumulation of microtubules
at one side of the nucleus in the region called the microtubule
organizing center (MTOC). Group I contains compounds 15 and 19
that exert a mild effect: the treated cells show a well organized
network but a lack of a perinuclear region enriched in microtu-
bules. Group II contains compounds 14, 16 and 17 that induce
severe effects on microtubule arrangement: about 50% of the cells
become roundish, whereas the cells showing a conventional
morphology lack microtubules at the cell periphery and display
In this paper the preparation of homo- and heterodimers of
podophyllotoxin and thiocolchicine is described, along with their
biological evaluation in a number of cellular assays. Thiocolchicine
homodimers exhibited a much greater ability to disrupt tubulin
polymerization in vitro than dimers containing podophyllotoxin,
but this was not directly reflected in their in vivo activity, with
compounds based on both parent molecules (particularly
compounds 12, 13, 18 and 20) acting as potent disruptors of the
microtubule network in A549 cells. The nature of the linker unit can
have an important influence on the biological activity of the dimer.

D. Passarella et al. / European Journal of Medicinal Chemistry 45 (2010) 219–226
223
The presence of two amino groups on the spacer improves the
water solubility but reduces the biological efficacy, while the
potential cleavage of disulfide-linked dimers by the high levels of
glutathione in tumour cells may result in these compounds acting
in their monomeric form. In future research we will be examining
the potential of these hybrid compounds to overcome drug resis-
tance frequently encountered with chemotherapeutic agents.
of H2 for 4 h. The reaction mixture was filtered through celite to
remove the catalyst and the solvent was removed in vacuo to give
compound 9 (118 mg, 98%). 1H NMR (400 MHz, CDCl3)
d 8.82 (br s,
2H), 5.17–5.41 (m, 2H), 4.31–4.40 (m, 2H), 1.74–1.80 (m, 4H), 1.60–
1.73 (m, 4H), 1.42 (s, 18H); 13C NMR (100 MHz, CDCl3) detected
signals
d 176.9, 155.8, 80.3, 53.2, 32.1, 28.3, 27.8. ESI positive MS:
Anal. Calcd. for C18H32N2O8Na 427.2051; found 427.2046.
4. Material and methods
4.5. General procedure for the preparation of compounds 12–19
4.1. N-Boc-
L
-allylglycine
Method A: A mixture of dicarboxylic acid (0.20 mmol), DMAP
(0.20 mmol), DCC (0.60 mmol) and deacetylthiocolchicine or
podophyllotoxin (0.40 mmol) in CH2Cl2 (10 mL) was stirred at room
temperature (19–60 h). The mixture was submitted to column
L-Allylglycine (250 mg; 2.17 mmol) was dissolved in 10 mL of
a mixture dioxane/water 1:1. (Boc)2O (980 mg; 2.39 mmol) was
added and the reaction mixture was stirred for 18 h. The organic
component of the solvent of the reaction was removed in vacuo and
the aqueous residue was acidified with HCl until pH 4. The solution
was extracted with Et2O and the combined organic phases were
dried over sodium sulfate. After evaporation of the solvent, N-Boc-
chromatography. Method B:
A mixture of dicarboxylic acid
(0.20 mmol), DMAP (0.20 mmol), DCC (0.60 mmol) and deace-
tylthiocolchicine or podophyllotoxin (0.40 mmol) was stirred at
50 ꢁC for 4 h in THF (3 mL). The mixture was submitted to column
chromatography.
L
-allylglycine was obtained as a colorless oil (444 mg; 95%). 1H NMR
(400 MHz, CDCl3) 8.86 (br s, 1H), 5.75–5.70 (m, 1H), 5.14–5.19 (m,
3H), 4.43–4.39 (m, 1H), 2.59–2.55 (m, 2H), 1.44 (s, 9H). 13C NMR
(100 MHz, CDCl3) detected signals 176.0, 155.4, 132.1, 119.1, 80.1,
d
4.6. Acetamide 2,20-dithiobis[N-(N-deacetylthiocolchicinyl)] 12
d
[19] Method A. Deacetylthiocolchicine and dithiodiglycolic acid
52.7, 36.3, 28.1. Anal. Calcd. for C10H17NO4: C, 55.80; H, 7.96; N, 6.51;
found: 55.83; H, 7.92; N, 6.52.
4. Column chromatography (CH2Cl2/EtOH 27/1) afforded
compound 12 as a yellow amorphous solid in 67% yield; [
0.64 (CHCl3, c ¼ 1). Anal. Calcd. for C44H48N2O10S4: C, 59.17; H, 5.42;
a
]D: þ
4.2. (2S)-Benzyl 2-(tert-butoxycarbonylamino)pent-4-enoate 10
N, 3.14; found: 59.21; H, 5.44; N, 3.11.
Benzyl chloroformate (348
a solution of N-Boc- -allylglycine (444 mg; 2.06 mmol), trietha-
nolamine (355 g; 2.38 mmol) and DMAP (25 mg; 0.20 mmol) in
m
L; 2.47 mmol) was added at 0 ꢁC to
4.7. Propanamide 3,30-dithiobis[N-(N-deacetylthiocolchicinyl)] 13
L
m
[19] Method A. Deacetylthiocolchicine and 3,30-dithiodipro-
pionic acid 5. Column chromatography (CH2Cl2/MeOH 20/1)
afforded compound 13 as a yellow amorphous solid in 50% yield;
CH2Cl2 (6 mL). After 2 h the reaction mixture was washed with
NaHCO3 (5%) and HCl (1 M). The aqueous layers were extracted
with CH2Cl2, and the combined organic phases were dried over
sodium sulfate and the solvent removed in vacuo. The crude residue
was submitted to column column chromatography (EtOAc/hexane
1:10) to give 540 mg of the desired product as a colorless oil (86%).
[
a
]D: - 0.18 (CHCl3, c ¼ 1); Anal. Calcd. for C46H52N2O10S4: C, 59.98;
H, 5.69; N, 3.04; found: C, 59.93; H, 5.72; N, 3.02.
4.8. 2-Aminopropanamide 3,30-dithiobis[N-(N-
deacetylthiocolchicinyl)] 14
1H NMR (400 MHz, CDCl3)
5.00–5.25 (m, 5H), 4.37–4.49 (m, 1H), 2.43–2.61 (m, 2H), 1.44 (s,
9H). 13C NMR (100 MHz, CDCl3) detected signals
171.9, 155.2,
d
7.33–7.39 (m, 5H), 5.61–5.71 (m, 1H),
d
Condensation of deacetylthiocolchicine and (Boc–Cys–OH)2 8
135.4, 132.2, 128.5, 128.4, 128.3, 119.1, 79.9, 67.0, 53.0, 36.7, 28.3. ESI
positive MS: Anal. Calcd. for C17H23NO4Na: 328.1519; found
328.1518.
according Method B afforded the corresponding Boc-protected
dimer (82.7 mg; 0.07 mmol). The crude product was dissolved in
dioxane (5 mL) and treated with a saturated solution of HCl in
dioxane (5 mL) at ꢂ10 ꢁC. The solution was stirred at 0 ꢁC for 1.5 h,
before the solvent was removed in vacuo and the resulting yellow–
orange solid was dissolved in CH2Cl2 and washed with a saturated
solution of NaHCO3. Removal of the solvent in vacuo gave
compound 14 as a yellow amorphous solid (63.7 mg; 95% yield). Rf:
4.3. (2S,7S)-Dibenzyl 2,7-bis(tert-butoxycarbonylamino)
oct-4-enoate 11
Compound 10 (100 mg; 0.32 mmol) was dissolved in dry
toluene (6 mL) under an inert atmosphere (N2). Grubbs’ catalyst
(II generation) (14 mg; 0.016 mmol) was added and the mixture
was heated to 55 ꢁC for 16 h. The solvent was removed in vacuo and
the residue was submitted to column column chromatography
(EtOAc/hexane 1:10) to give 60 mg of the desired product as
0.18 (CH2Cl2/MeOH 10/1). [
a
]D: - 1.5 (CHCl3, c ¼ 0.4). 1H NMR
(CDCl3, 300 MHz, detected signals) 7.30 (2H, d, J ¼ 6), 7.20 (2H, d,
J ¼ 6), 6.80 (2H, s), 4.50–4.40 (2H, m) 3.88 (6H, s), 3.85 (6H, s), 3.62
(6H, s), 2.45 (6H, s), 2.40–2.25 (2H, m) 1.82–1.71 (4H, m); 13C NMR
(CDCl3, 100 MHz) 182.0, 171.7, 157.3, 154.1, 151.1, 143.8, 138.7, 136.0,
131.1, 126.6, 126.1, 125.2, 108.2, 61.7, 60.0, 56.0, 54.2, 38.4, 36.5, 30.6,
15,1; Anal. Calcd. for C46H54N4O10S4: C, 58.08; H, 5.72; N, 5.89;
found: C, 58.07; H, 5.70; N, 5.88. Data for the Boc-protected dimer:
Rf: 0.22 (CH2Cl2/AcOEt 1.5/1); aD: – 6.00 (CHCl3, c ¼ 1). 1H NMR
(CDCl3, 300 MHz, detected signals) 8.60–8.50 (2H, m), 7.60 (2H, s),
7.3–7.25 (2H, d, J ¼ 11), 7.07–7.02 (2H, d, J ¼ 11), 6.55 (2H, s), 4.80–
4.71 (4H, m) 3.93 (6H, s), 3.90 (6H, s), 3.60 (6H, s), 2.40 (6H, s), 1.40
a colorless oil (yield: 64%). 1H NMR (400 MHz, CDCl3)
d 7.29–7.39
(m, 10H), 5.25–5.34 (m, 2H), 5.04–5.24 (m, 6H), 4.31–4.40 (m, 2H),
2.32–2.51 (m, 4H), 1.43 (s, 18H). 13C NMR (100 MHz, CDCl3) detected
signals
d 171.7, 155.1, 135.4, 128.6, 128.4, 79.9, 67.0, 53.1, 35.4, 28.3;
ESI positive MS: Anal. Calcd. for C32H42N2O8Na 605.2833; found
605.2825.
4.4. (2S,7S)-2,7-Bis(tert-butoxycarbonylamino)octanoic acid 9
(18H, s); 13C NMR (CDCl3, 100 MHz) detected signals
d 182.3, 169.3,
158.1, 156.6, 154.8, 151.1, 141.8, 138.0, 135.0, 131.5, 128.5, 126.0, 125.5,
107.4 79.0, 61.5, 60.0, 56.1, 54.0, 38.3, 36.7, 30.6, 28.1, 15,1. Anal.
Calcd. for C56H70N4O14S4: C, 58.42; H, 6.13; N, 4.87; found: C, 58.45;
H, 6.10; N, 4.90.
Compound 11 (174 mg; 0.30 mmol) was dissolved in EtOAc
(10 mL) under an inert atmosphere (N2). Pd/C (50 mg, 5% w/w) was
added and the reaction mixture was stirred under one atmosphere

224
D. Passarella et al. / European Journal of Medicinal Chemistry 45 (2010) 219–226
4.9. 2,7-Diaminooctanediamide N-(N-deacetylthiocolchicinyl) 15
vacuo and the residue purified by column chromatography (CH2Cl2/
MeOH 15/1) to afford the product 18 as a white amorphous solid in
Condensation of deacetylthiocolchicine and (2S,7S)-2,7-bis(tert-
butoxycarbonylamino)-octanedioic acid 9 according to Method A
afforded the corresponding Boc-protected dimer (130 mg;
0.12 mmol). The crude product was dissolved in dioxane (5 mL) and
treated with a solution of HCl in dioxane (saturated, 5 mL) at
ꢂ10 ꢁC. The solution was then stirred at r.t. for 1 h. Removal of the
solvent in vacuo gave a yellow–orange amorphous solid that was
redissolved in CH2Cl2 (10 mL) and was washed with a saturated
solution of NaHCO3. The organic phase was dried with sodium
sulfate and the solvent removed in vacuo to afford a yellow solid
that was purified by column chromatography (CH2Cl2/MeOH 5/1)
21% yield. Rf: 0.17 (CH2Cl2/MeOH 15/1). [
a
]D: -1.3 (CHCl3, c ¼ 0.25).
1H NMR (CDCl3, 400 MHz, detected signals) 6.80 (2H, s), 6.60 (2H,
s), 6.45 (4H, s), 6.05 (4H, s), 6.03–5.98 (m, 2H), 4.75–4.65 (m, 2H)
4.63 (s, 2H), 4.42–4.38 (m, 2H), 4.22–4.19 (m, 2H) 3.70 (s, 12H),
3.35–3.15 (m, 2H), 2.90–2.80 (m, 4H); 13C NMR (CDCl3, 100 MHz)
detected signals
d 174.3, 172.3, 154.1(2C), 149.6, 148.5, 139.1, 135.5,
133.0, 128.0, 110.5, 109.5(2C), 108.0, 102.4, 76.0, 72.0, 61.5, 57.0, 46.1,
44.0, 39.3, 35.7; Anal. Calcd. for C50H52N2O18S2: C, 58.13; H, 5.07; N,
2.71; found: C, 58.15; H, 5.09; N, 2.68. Data for the Boc-protected
dimer: Rf: 0.45 (CH2Cl2/AcOEt 4/1);; [
a
]D: – 4.79 (CHCl3, c ¼ 1). 1H
NMR (CDCl3, 400 MHz, detected signals) 6.80 (s, 2H), 6.60 (s, 2H),
6.40 (s, 4H), 6.00 (s, 4H), 5.92–5.89 (m, 2H), 4.71 (s, 2H) 4.63 (s, 2H),
4.42–4.39 (m, 2H), 4.21–4.18 (m, 2H), 3.80 (s, 6H), 3.75 (s, 6H), 3.70
(s, 6H), 3.22–3.18 (m, 4H), 2.82–2.79 (m, 4H), 1.50 (s, 18H); 13C NMR
to give 15 as a yellow amorphous solid (111 mg; 99% yield). [a]D: –
3.2 (CHCl3, c ¼ 0.2). 1H NMR (400 MHz, CDCl3): 8.78 (d, J ¼ 7.6, 2H),
7.63 (s, 2H), 7.34 (d, J ¼ 10.4, 2H), 7.10 (d, J ¼ 10.8, 2H), 6.53 (s, 2H),
4.68–4.80 (m, 2H), 3.94 (s, 6H), 3.90 (s, 6H), 3.68 (s, 6H), 2.46–2.55
(m, 2H), 2.42 (s, 6H), 2.20–2.39 (m, 8H), 1.74–2.15 (m, 6H). 13C NMR
(CDCl3, 100 MHz) detected signals
d 174.3, 172.3, 154.1(2C), 149.6,
148.8, 139.1, 135.5, 133.0, 128.0, 110.5, 109.5(2C), 108.0, 102.4,
78.0(2C), 76.0, 72.0, 61.5, 57.0, 46.1, 44.0, 39.3, 35.7, 30.6, 29.1. Anal.
Calcd. for C60H68N2O22S2: C, 58.43; H, 5.56; N, 2.27; found: 58.45; H,
5.59; N, 2.30.
(100 MHz, CDCl3) detected signals
d 182.1, 173.7, 158.3, 153.7, 152.8,
151.1, 141.6, 139.1, 134.9, 134.6, 129.1, 127.0, 125.6, 107.5, 61.7, 61.4,
56.1, 54.0, 52.3, 36.3, 33.7, 30.1, 24.2, 15.1. ESI positive MS: Anal.
Calcd. for C48H58N4O10S2Na: 937.3487; found: 937.3465. Data for
the Boc-protected dimer: 1H NMR (400 MHz, CDCl3): 8.60 (d,
J ¼ 7.6, 2H), 7.85 (s, 2H), 7.32 (d, J ¼ 10.4, 2H), 7.08 (d, J ¼ 10.4, 2H),
6.52 (s, 2H), 5.39 (d, J ¼ 7.2, 2H), 4.79 (dt, J ¼ 10.8, 7.4 Hz, 2H), 4.48
(dd, J ¼ 6.4, 4.8 Hz, 2H), 3.94 (s, 6H), 3.89 (s, 6H), 3.69 (s, 6H), 2.46–
2.54 (m, 2H), 2.41 (s, 6H), 2.14–2.40 (m, 8H), 1.86–2.07 (m, 6H), 1.37
4.13. Dipodophyllotoxin-4-yl adipate 19
Method A: podophyllotoxin 2 and adipic acid 7. Column chro-
matography (CH2Cl2/AcOEt 50/1) afforded compound 19 as a white
amorphous solid in 44% yield; [
a
]D: – 0.7 (CHCl3, c ¼ 0.4). 1H NMR
(s, 18H).13C NMR (100 MHz, CDCl3) detected signals
d
181.9, 171.2,
(400 MHz, CDCl3): 6.74 (s, 2H), 6.53 (s, 2H), 6.38 (s, 4H), 5.95 (s, 4H),
5.86 (d, J ¼ 8.8, 2H), 4.59 (d, J ¼ 4.4, 2H), 4.33 (dd, J ¼ 9.2, 6.8 Hz,
2H), 4.13 (t, J ¼ 9.8, 2H), 3.78 (s, 6H), 3.74 (s,12H), 2.89–2.97 (m, 2H),
2.75–2.87 (m, 2H), 2.42–2.51 (m, 4H), 1.71–1.78 (m, 4H). 13C NMR
158.5, 155.3, 153.6, 152.7, 151.1, 141.6, 138.9, 134.7,129.7, 126.8, 125.8,
107.5, 79.0, 61.7, 61.3, 56.1, 53.4, 52.3, 36.2, 32.5, 30.2, 28.3, 23.6,
15.1. ESI positive MS: Anal. Calcd. for C58H74N4O14S2Na: 1137.4535;
found: 1137.4496.
(100 MHz, CDCl3) detected signals d 173.6, 173.5, 152.6, 148.1, 147.5,
137.1, 134.8, 132.4, 128.2, 109.7, 108.1, 106.9, 101.5, 73.7, 71.3, 60.7,
56.1, 45.5, 43.7, 38.6, 33.8, 24.2. ESI positive MS: Anal. Calcd. for
C50H50O18Na: 961.2889; found: 961.2864
4.10. Diadipamide N-(N-deacetylthiocolchicinyl) 16
Method A. Deacetylthiocolchicine and adipic acid 7. Column
chromatography (CH2Cl2/MeOH 40/1) afforded compound 16 as
4.14. Podophyllotox-4-yl 4-[N-(N-deacetylthiocolchicinyl)-4-
a yellow amorphous solid in 45% yield. [
a
]D: – 1.2 (CHCl3, c ¼ 0.3).
oxobutyl disulfanyl butanoate 20
1H NMR (300 MHz, CDCl3): 8.85 (d, J ¼ 6, 2H), 7.71 (s, 2H), 7.35 (d,
J ¼ 10, 2H), 7.12 (d, J ¼ 10, 2H), 6.57 (s, 2H), 4.66 (ddd, J ¼ 12, 6, 6,
2H), 3.92 (s, 6H), 3.90 (s, 6H), 3.65 (s, 6H), 2.60–2.65 (m, 2H), 2.45 (s,
6H), 1.70–2.40 (m, 14H).13C NMR (75 MHz, CDCl3) detected signals
A solution of 4,40-dithiodibutyric acid 6 (198 mg; 0.83 mmol),
DMAP (38 mg; 0.31 mmol), DCC (194 mg; 0.94 mmol) and deace-
tylthiocolchicine (234 mg; 0.63 mmol) was stirred at room
temperature for 2 h before being filtered through celite. The solvent
was removed in vacuo and the residue was dissolved in CH2Cl2 and
washed with a solution of ammonium hydroxide. The organic
phase was dried over sodium sulfate and the solvent was removed
in vacuo to give the corresponding crude monomer (265 mg, 65%
yield). The monomer (178 mg, 0.30 mmol), DMAP (26 mg,
0.21 mmol), DCC (123 mg, 0.60 mmol) and podophyllotoxin 2
(124 mg, 0.30 mmol) were dissolved in CH2Cl2 (10 mL). After 7 h
the solvent was removed in vacuo and the residue purified by
column chromatography (CH2Cl2/MeOH 5/1) to give 20 (253 mg,
86% yield) as a yellow amorphous solid. Rf: 0.59 (CH2Cl2/MeOH 30/1).
d
181.9, 173.1, 158.0, 153.7, 152.7, 151.0, 141.0, 139.2, 134.9, 134.7,
128.6, 127.0, 125.8, 107.4, 61.8, 61.4, 56.1, 53.0, 37.0, 35.1, 30.1, 25.8,
15.1; Anal. Calcd. for C46H52N2O10S2: C, 64.47; H, 6.12; N, 3.27.
Found: C, 64.51; H, 6.13; N, 3.29.
4.11. Dipodophyllotoxin-4-yl 2,20-dithiobisacetate 17
[19] Method A. Podophyllotoxin 2 and dithiodiglycolic acid 4.
Column chromatography (CH2Cl2/MeOH 20/1) afforded compound
17 as a white amorphous solid in 50% yield. Rf: 0.53 (CH2Cl2/MeOH
20/1); [
a
]D: – 0.53 (CHCl3, c ¼ 1). Anal. Calcd. for C48H46O18S2: C,
59.13; H, 4.76. Found: C, 59.16; H, 4.78.
[a
]D: – 0.9 (CHCl3, c ¼ 0.5). 1H NMR (CDCl3, 300 MHz, detected
signals): 8.52 (s, 1H, Thio), 7.25 (d, J ¼ 9.4, 1H, Thio), 7.17 (s, 1H,
Thio), 7.03 (d, J ¼ 9.4, 1H, Thio), 6.75 (s, 1H, Pod), 6.53 (s, 1H, Thio),
6.51 (s, 2H, Pod), 6.38 (s, 2H, Pod), 5.98 (d, J ¼ 7.5, 2H, Pod), 5.88 (d,
J ¼ 9.4, 1H, Pod), 4.68–4.56 (2H, m, Thio þ Pod), 4.36 (dd, J ¼ 6.9, 9.7,
1H, Pod), 4.21 (dd, J ¼ 3.2, 6.5, 1H, Pod), 3.91 (s, 3H, Thio), 3.89 (s,
3H, Thio), 3.81 (s, 3H, Pod), 3.75 (s, 6H, Pod), 3.63 (s, 3H, Thio), 2.97–
2.94 (m, 1H, Pod), 2.85–2.75 (m, 1H, Pod), 2.76–2.49 (m, 2H, Thio),
2.42 (s, 3H, Thio), 2.41–1.86 (m, 14H, Thioþ linker). 13C NMR
4.12. Dipodophyllotoxin-4-yl 3,30-bisthio-2-amino propanoate 18
Condensation of podophyllotoxin
2 and (Boc–Cys–OH)2 8
according Method B afforded the corresponding Boc-protected
dimer (149.3 mg; 0.12 mmol). The crude product was dissolved in
CH2Cl2 (10 mL), TFA (2.22 g; 1.5 mL; 19.4 mmol) was added at
ꢂ15 ꢁC and the reaction mixture was stirred for 48 h. The organic
phase was washed with a saturated solution of NaHCO3 at ꢂ20 ꢁC.
The temperature was allowed to rise to r.t. over 1 h. The organic
phase was dried over sodium sulfate, the solvent was removed in
(100 MHz, CDCl3) detected signals
d 182.6 (1C, Thio), 173.5 (1C,
Podo), 170.0 (1C, Linker), 168.5 (1C, Linker), 158.3 (1C, Thio), 153.5
(1C, Thio), 152.1 (2C, Podo), 151.9 (1C, Thio), 151.0 (1C, Thio), 148.4

D. Passarella et al. / European Journal of Medicinal Chemistry 45 (2010) 219–226
225
(1C, Podo), 147.7 (1C, Podo), 141.7 (1C, Thio), 138.5 (1C, Thio), 137.4
(1C, Podo), 135.1 (1C, Thio), 133.9 (1C, Podo), 133.2 (1C, Thio), 130.4
(1C, Podo), 128.1 (1C, Thio),127.3 (1C, Podo), 126.1 (1C, Thio), 126.0
(1C, Thio), 109.2 (1C, Podo), 108.3 (2C, Podo), 107.5 (1C, Thio), 107.0
(1C, Podo), 101.1 (1C, Podo), 74.0 (1C, Podo), 71.0 (1C, Podo), 61.4
(1C, Thio), 61.2 (1C, Thio), 60.1 (1C, Podo), 56.0 (1C, Thio), 55.9 (2C,
Podo), 52.1 (1C, Thio), 45.5 (1C, Podo), 44.3 (1C, Podo),42.1 (1C,
Thio), 38.1 (1C, Podo), 36.5 (1C, Linker), 34.3 (2C, Linker), 34.0 (2C,
Linker), 33.6 (1C, Linker), 30.1 (1C, Thio), 15.0 (1C, Thio). Anal. Calcd.
for C50H55NO14S3: C, 60.65; H, 5.60; N,1.41; found: C, 60.68; H, 5.62;
N, 1.43.
K-Pipes pH 6.9, 2 mM EGTA, 1 mM MgCl2, 10% glycerol). The reac-
tion mixtures were incubated at 37 ꢁC for 15 min to allow slow
binding drugs to bind to the tubulin. The reaction mixtures were
then chilled on ice, GTP (final concentration, 1 mM) was added, and
they were incubated for 30 min at 37 ꢁC. At the end of polymeri-
zation, unpolymerized and polymerized fractions of tubulin were
separated by centrifugation at 16500 ꢃ g for 30 min at 30 ꢁC. The
collected microtubules were resuspended in SDS-PAGE sample
buffer (2% w/v SDS, 10% v/v glycerol, 5% v/v b-mercaptoethanol,
0.001% w/v bromophenol blue, and 62.5 mM Tris, pH 6.8) and the
unpolymerized tubulin was diluted 3:1 with 4X SDS-PAGE sample
buffer. Equal proportions of each fraction were resolved by a 7.5%
SDS-gel and stained with Coomassie blue. Densitometric analyses
of stained gels was performed by using the ImageMaster VDS
Software (Pharmacia Biotech), and the obtained data were elabo-
rated using SigmaPlot 8.0 program (Systat Software Inc., Point
Richmond, CA, USA). At least three independent experiments were
performed with each compound. Differences between the effects of
the different compounds were evaluated by ANOVA followed by
Dunnet’s test.
4.15. In vitro activity with
L-glutathione
L-Glutathione (0.009 mmol) in water was added to a solution of
12 (0.009 mmol) in acetone (5 mL) at room temperature. After 6 h
the reaction mixture was analyzed by HRESI-MS. GS-SG and Thio–
CO–CH2–S–S–G were detected. HRESI positive MS: GS-SG Anal.
Calcd. for C20H32N6O12S2Na: 635.1415; found: 635.1417 ESI positive
MS: Thio–CO–CH2–S–S–G Anal. Calcd. for C32H40N4O11S3Na:
775.1748; found: 775.1745.
4.18. Cytotoxicity by MTS assay
4.16. Immunofluorescence analyses
Cells were harvested and plated in 96-well flat-bottomed
microplates at a density of 103 cells/well. Assays were performed in
quintuplicates. Cells were allowed to attach for 24 h. The drugs
Microtubule organization in cell was revealed by indirect
immunofluorescence (IF) analyses. Human lung carcinoma cell line
A549 (CCL-185; American Type Culture Collection, Rockville, MD,
U.S.A.) was grown in minimal essential medium with Earle’s
(E-MEM), supplemented with 10% fetal bovine serum (Hyclone
were prepared in medium at different concentrations (0.1
and 10 M) and were added to the plates at a volume of 100
After 24 (or 96) h incubation 20
l of the CellTiter 96Ò AQueous One
mM, 1
mM
m
ml/well.
m
Europe, Oud-Beijerland, Holland), 2 mM
L
-glutamine, 100 U/ml
Solution Reagent (Promega Corporation, Madison, WI, USA) were
added to each well and the plates were incubated for one hour at
37 ꢁC. The CellTiter 96Ò AQueous One Solution Reagent contains
a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-car-
penicillin and non-essential amino acids. Cells were maintained at
37 ꢁC in a humidified atmosphere at 5% CO2. Experiments were
carried out with cells plated on glass coverslips at a density of
1.5 ꢃ104 cells/cm2 and grown for 24 h in control medium following
boxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,
inner
an incubation of 24 h in the presence of 0.5
mM of the drug or
salt; MTS] and an electron coupling reagent (phenazine etho-
sulfate; PES). PES has a high chemical stability, which allows it to
form a stable solution with MTS. The absorbance was read at
490 nm on a plate spectrophotometer (Victor3Ô 1420 Multilabel
Counter, Perkin Elmer Instruments, Shelton, USA). Cell cytotoxicity
was expressed as the percentage of the controls.
solvent vehicle alone (DMSO). At the end of the treatments, cells
were fixed and stained as previously described. [22] Briefly, A549
cells were fixed and permeabilized for 10 min with methanol at
ꢂ20 ꢁC, washed with PBS and blocked in PBS þ 1% bovine serum
albumin (BSA) for 15 min at room temperature. To localize tubulin,
the cells were incubated with monoclonal anti a-tubulin antibody
(clone B-5-1-2, Sigma–Aldrich), 1:500 in PBS for 1 h at 37 ꢁC. As
secondary antibodies we used goat anti-mouse Alexa FluorÔ 594
(Molecular Probes), 1:1000 in PBS þ 5% BSA for 45 min at 37 ꢁC.
Acknowledgments
This research has been developed under the umbrella of CM
0602 COST Action "Inhibitors of Angiogenesis: design, synthesis and
biological exploitation’’.
Nuclei staining was performed by incubation with DAPI (0.25 mg/ml
in PBS) for 15 min at room temperature. The coverslips were
mounted in MowiolÒ (Calbiochem)–DABCO (Sigma–Aldrich) and
examined with a Zeiss Axiovert 200 microscope equipped with
a 63 ꢃ Neofluor lens. Images were acquired with an Axiocam
camera (Zeiss) and PC running Axiovision software (Zeiss).
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[20] Data for compound 21: 1H NMR (400 MHz, Acetone-d6): 8.60–8.50 (m, 3H, NH),
7.7.27–7.13 (m, 3H, Thio), 6.78 (s, 1H, Podo), 6.53 (s, 1H, Thio), 6.47 (s, 1H, Podo),
6.38 (s, 2H, Podo), 6.05 (d, J ¼ 8.5, 2H, Podo), 5.92 (d, J ¼ 8.3, 1H, Podo), 4.71 (d,
J ¼ 7.4, 1H, Podo), 4.63–4.51 (m, 3H, ThioþLinker), 4.43 (dd, J ¼ 12.1, 8.2, 1H,
Podo), 4.253 (t, J ¼ 10.7,1H, Podo), 3.94 (s, 3H, Thio), 3.89 (s, 3H, Thio), 3.68 (s, 9H,
ThioþPodo), 3.59 (s, 3H,Podo), 3.30–3.20 (m, 2H, CH2Linker), 3.05–2.93 (m, 2H,
Podo) 2.71–2.62 (m, 2H, CH2Linker), 2.50–2.41 (m, 2H, Thio), 2.45 (s, 3H, Thio),
2.09–1.75 (m, 2H, Thio), 1.50 (s, 9H, Boc), 1.43 (s, 9H, Boc).13C NMR (100 MHz,
Acetone-d6): 182.2 (1C, Thio), 174.8 (1C, Podo), 171.0 (1C, Linker), 169.1 (1C,
Linker), 161.3 (2C, Boc), 158.2 (1C, Thio), 157.9 (1C, Boc), 153.8 (1C, Thio), 152.7
(2C, Podo), 152.2 (1C, Thio), 151.1 (1C, Thio), 148.5 (1C, Podo), 148.0 (1C, Podo),
141.6 (1C, Thio), 138.7 (1C, Thio), 137.6 (1C, Podo), 135.5 (1C, Thio), 134.5 (1C,
Podo), 134.1 (1C, Thio), 131.1 (1C, Podo), 128.7 (1C, Thio), 128.3 (1C, Podo), 127.1
(1C, Thio), 125.9 (1C, Thio), 109.2 (1C, Podo), 108.3 (2C, Podo), 107.7 (1C, Podo),
107.3 (1C, Thio), 101.6 (1C, Podo), 79.1(2C, Boc), 74.1 (1C, Podo), 70.8 (1C, Podo),
64.9 (1C, Thio), 60.7 (1C, Thio), 60.3 (1C, Podo), 59.6 (1C, Thio), 55.5 (2C, Podo),
53.9 (1C, Linker), 53.8 (1C, Thio), 52.1 (1C, Thio), 44.6 (1C, Podo), 43.7 (1C, Podo),
41.3 (1C, Thio), 38.6 (1C, Podo), 35.7 (1C, Linker), 34.6 (1C, Linker), 29.8 (1C,
Thio), 29.0 (3C, Boc), 28.4 (3C, Boc), 15.1 (1C, Thio); Anal. Calcd. for
C58H69N3O18S3: C, 58.42; H, 5.83; N, 3.52. Found: C, 58.44; H, 5.82; N, 3.53
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